Immunoassays for high positively charged proteins

Patent metadata

Field	Value
Publication number	US-12474329-B2
Application number	US-202318164275-A
Country	US
Kind code	B2
Filing date	Feb 3, 2023
Priority date	Jan 29, 2015
Publication date	Nov 18, 2025
Grant date	Nov 18, 2025

Abstract

The present invention relates to immunoassays for quantification of a high positively charged protein, such as a FGF-18 protein, in human synovial fluid sample.

First claim

The invention claimed is:

1 . A method for quantification of a highly positively charged protein in a human synovial fluid sample comprising the steps of:
a) pre-treating the human synovial fluid sample, the pre-treating step comprising adding hyaluronidase solution to the human synovial fluid sample, incubating said sample at room temperature (RT), and centrifuging the human synovial fluid sample; b) diluting the pre-treated human synovial fluid sample with a buffer; c) immobilizing a biotinylated antibody against the highly positively charged protein to a column; d) washing the column to remove unbound antibody with a standard wash buffer; e) contacting in the column the pre-treated and diluted human synovial fluid sample with the immobilized biotinylated antibody under conditions in which the antibody binds specifically to the highly positively charged protein, to produce an antibody-protein complex; f) washing the column with a standard wash buffer; g) adding to the antibody-protein complex in the column a fluorescent dye-labelled antibody specific for the highly positively charged protein to produce a measurable response, and washing the column with a standard wash buffer; h) measuring the response produced; and i) determining a quantity of highly positively charged protein in the sample by comparing the response produced with the sample to the response produced with a calibration standard; wherein the highly positively charged protein in the sample is fibroblast growth factor 18 (FGF-18) having an isoelectric point at or above 9.5, and wherein the FGF-18 protein is selected from the group consisting of: a) a polypeptide comprising the amino acid residues 28-207 of SEQ ID NO: 1, b) a polypeptide comprising the amino acid residues 28-196 of SEQ ID NO: 1, and c) a polypeptide comprising SEQ ID NO: 2.

2 . The method according to claim 1 , wherein the incubating time of step a) is 1 h.

3 . The method according to claim 1 , wherein the FGF-18 protein is sprifermin.

4 . A method for automatic quantification of a highly positively charged protein in a human synovial fluid sample comprising the steps of:
a) pre-treating the human synovial fluid sample, the pre-treating step comprising adding hyaluronidase solution to the human synovial fluid sample, incubating said sample at room temperature (RT), and centrifuging the human synovial fluid sample; b) diluting the pre-treated human synovial fluid sample with a buffer; c) immobilizing a biotinylated antibody against the highly positively charged protein to a column; d) washing the column to remove unbound antibody with a standard wash buffer; e) providing an injection means for automatic transfer of the pre-treated and diluted human synovial fluid sample to the column; f) washing the injection means with a high ionic force buffer that is 1.5M NaCl in 20% ethanol before the pre-treated and diluted human synovial fluid sample is transferred to the column; g) transferring the pre-treated and diluted human synovial fluid sample to the column, thereby contacting the pre-treated and diluted human synovial fluid sample with the immobilized biotinylated antibody under conditions in which the antibody binds specifically to the highly positively charged protein, to produce an antibody-protein complex; h) washing the injection means with the high ionic force buffer after the step g); i) washing the column with a standard wash buffer; j) adding to the antibody-protein complex in the column a fluorescent dye-labelled antibody specific for the highly positively charged protein to produce a measurable response, and washing the column a standard wash buffer; k) measuring the response produced; and l) determining a quantity of the highly positively charged protein in the sample by comparing the response produced with the sample to the response produced with a calibration standard; wherein the highly positively charged protein in the sample is fibroblast growth factor 18 (FGF-18) having an isoelectric point at or above 9.5, and wherein the FGF-18 protein is selected from the group consisting of: a) a polypeptide comprising the amino acid residues 28-207 of SEQ ID NO:1, b) a polypeptide comprising the amino acid residues 28-196 of SEQ ID NO:1, and c) a polypeptide comprising SEQ ID NO:2.

5 . The method according to claim 4 , wherein the human synovial fluid sample is part of a set of human synovial fluid samples to be analyzed.

6 . The method according to claim 4 , wherein the column of step c) is one column in a set of columns.

7 . The method according to claim 4 , wherein all the steps are repeated as often as needed to automatically quantify a high positively charged protein in a set of human synovial fluid samples to be analyzed.

8 . The method according to claim 4 , wherein the incubating time of step a) is 1 h.

9 . The method according to claim 4 , wherein the FGF-18 protein is sprifermin.

Assignees

• Ares Trading Sa

Inventors

• Bertotti Elisa
• Castagna Valeria

Classifications

• G01N2333/50

  Fibroblast growth factors [FGF] · CPC title

• G01N2035/00495

  Centrifuges · CPC title

• G01N35/1095

  for supplying the samples to flow-through analysers (for a specific analyser see relevant groups, e.g. under G01N15/00, G01N21/00, G01N27/00, G01N30/00, H01J49/00) · CPC title

• G01N35/10

  Devices for transferring samples {or any liquids} to, in, or from, the analysis apparatus, e.g. suction devices, injection devices {(G01N35/0099 takes precedence)} · CPC title

• G01N35/0099

  comprising robots or similar manipulators (robots per se B25J) · CPC title